This invention relates to compositions useful in the generation of an immune response. The immune response includes, inter alia, provocation of T cells, such as cytolytic T cells, against complexes of a p53 derived peptide and MHC molecules or a p53 derived peptide or protein itself, as well as antibodies against such peptides and proteins. Such T cells and antibodies may be generated, e.g., in a mouse, rat, rabbit, sheep, goat or other non-human animal, and then used in diagnostic methods to identify tumor presence. They may also be generated in vitro such as by cell culture and hybridoma techniques. The compositions may also be used, therapeutically, via administration to a subject afflicted with a cancerous condition or one where cell transformation has taken place, to provoke an immune response against tumors, cancer cells, and transformed cells.
Adjuvants, broadly defined, are substances which promote immune responses. Frequently, the adjuvant of choice is Freund""s complete adjuvant, or killed B. pertussis organisms, used in combination with alum precipitated antigen. A general discussion of adjuvants is provided in Goding, Monoclonal Antibodies:
Principles and Practice (Second edition, 1986), at pages 61-63, which are incorporated by reference herein. Goding notes, however, that when the antigen of interest is of low molecular weight, or is poorly immunogenic, coupling to an immunogenic carrier is recommended. Such molecules, according to Goding, generally have molecular weights below about 1000. Among the carriers suggested by Goding, at page 283, are keyhole limpet hemocyanin, bovine serum albumin, ovalbumin, and fowl immunoglobulin.
What is problematic about such carriers, however, is that frequently they are also immunogenic themselves. Thus, the immune response may be a general one, with part, most, or all of it being directed against the carrier molecule rather than the immunogen itself.
Exemplary of developments in the art as they relate to adjuvants is U.S. Pat. No. 5,057,540 to Kensil, et al, the disclosure of which is incorporated by reference herein. Kensil et al disclose the preparation of various saponin extracts, which are useful as adjuvants in immunogenic compositions. As they are natural products, the extracts are not completely defined. Kensil, et al do provide a complete and enabling disclosure for how various extracts, including QA-7, QA-19, and QA-21 (also referred to as QS-21) are prepared. Experiments are set forth in which bovine serum albumin (xe2x80x9cBSAxe2x80x9d) was combined with various extracts (examples 8 and 9), and where feline leukemia virus recombinant glycoprotein xe2x80x9cgp7ORxcex94 was tested, following absorption to aluminum hydroxide (alum). The two immunogens tested, however, are expected to be immunogenic in their own right (gp7 ORxcex94 has a molecular weight of 70 kd, and serum albumin has about the same molecular weight). No experiments were carried out at all on molecules which should, per se, be considered to be poorly or even non-immunogenic, and thus would be expected to require the use of alum absorption or the use of haptenic carriers for provocation of a response.
In PCT Application WO9219758, which corresponds to defensive publication 7697275, which is incorporated by reference herein, an adjuvant referred to as xe2x80x9cMTP-MF59xe2x80x9d is disclosed. This adjuvant is used in connection with a Plasmodium falciparum protein, xe2x80x9cPfs-25-Bxe2x80x9d. This combination is described as a transmission blocking vaccine. The P. falciparum protein is itself large enough to be immunogenic. Thus, none of the art shows that the improved adjuvants can be used in combination with presumptively non-immunogenic proteins and peptides to yield immunologically effective compositions.
Interleukin-12, or xe2x80x9cIL-12xe2x80x9d hereafter, is known to play an important role in the differentiation of TH0-TH1, and TH2-TH0 cells in vivo. See Manetti, et al, J. Exp. Med 177:1199-1204 (1993); Hsieh, et al, Science 260:547-549 (1993).
The molecule referred to as p53 has been known for a number of years. A brief summary of the molecule is provided by Culotta, et al., Science 262: 1958-1961 (1993) (xe2x80x9cp53 Sweeps Through Cancer Researchxe2x80x9d). As reported therein, about 50% of all subjects diagnosed with cancer have a p53 mutation (or p53 mutations). These mutations can abolish the function of normal, or wild type p53, which is to act as a tumor suppressor of the observed mutations, about 90% involve a change in amino acid sequence (a xe2x80x9cmissensexe2x80x9d mutation), as compared to xe2x80x9cnonsensexe2x80x9d mutations, where the changes in the gene result in truncation or destabilization of the protein. The wild type p53 molecule is also involved in the cell cycle, in that it inhibits cell division. This is accomplished by activating a second protein of 21 kilodaltons, which inhibit the proteins xe2x80x9ccdkxe2x80x9d and xe2x80x9ccykxe2x80x9d, which are required for cell division.
It has now been found, surprisingly, that compositions can be made which comprise immunogenic peptides, such as p53 derived peptides, in combination with an adjuvant such as those described above and the cytokine interleukin-12, or xe2x80x9cIL-12xe2x80x9d. When administered to a subject animal, these compositions provoke an immunogenic response which is surprisingly strong, and totally unexpected in view of the response elicited with each element by itself, or pairs of these elements. In especially preferred embodiments the immunogenic peptide or protein is combined with the adjuvant QS21, which is disclosed in the Kensil, et al, patent, incorporated by reference supra.
A further aspect of this invention is the use of the cytokine, IL-12. Other interleukins, i.e., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, and IL-11, may also be used in view of their known efficacy as T-cell and B-cell growth factors. Of these, IL-2 is especially preferred. The compositions yield unexpected results when administered to T cells or B cells, in vitro or in vivo, in that their proliferation or other response, is surprisingly enhanced.
The immunogens of this invention include all p53 derived peptides and proteins, such as mutant p53 peptides, as exemplified.
The invention is described in greater detail in the disclosure which follows.